Characterization of a mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1 by biochemical, X-ray crystallographic, and mass spectrometric approaches.

We report the characterization of 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936) as a mechanism-based inhibitor of NQO1. Inactivation of NQO1 by ES936 was time- and concentration-dependent and required the presence of a pyridine nucleotide cofactor consistent with a need for metabolic activation. That ES936 was an efficient inhibitor was demonstrated in these studies by the low partition ratio (1.40 +/- 0.03). The orientation of ES936 in the active site of NQO1 was examined by X-ray crystallography and found to be opposite to that observed for other indolequinones acting as substrates. ES936 was oriented in such a manner that, after enzymatic reduction and loss of a nitrophenol leaving group, a reactive iminium species was located in close proximity to nucleophilic His 162 and Tyr 127 and Tyr 129 residues in the active site. To determine if ES936 was covalently modifying NQO1, ES936-treated protein was analyzed by electrospray ionization liquid chromatography/mass spectrometry (ESI-LC/MS). The control NQO1 protein had a mass of 30864 +/- 6 Da (n = 20, theoretical, 30868.6 Da) which increased by 217 Da after ES936 treatment (31081 +/- 7 Da, n = 20) in the presence of NADH. The shift in mass was consistent with adduction of NQO1 by the reactive iminium derived from ES936 (M + 218 Da). Chymotryptic digestion of the protein followed by LC/MS analysis located a tetrapeptide spanning amino acids 126-129 which was adducted with the reactive iminium species derived from ES936. LC/MS/MS analysis of the peptide fragment confirmed adduction of either Tyr 127 or Tyr 129 residues. This work demonstrates that ES936 is a potent mechanism-based inhibitor of NQO1 and may be a useful tool in defining the role of NQO1 in cellular systems and in vivo.

Differential involvement of caspases in hydroquinone-induced apoptosis in human leukemic hl-60 and jurkat cells.

The benzene metabolite hydroquinone (HQ) is postulated to exert its myelotoxicity by bioactivation to reactive quinone derivatives in myeloperoxidase (MPO)-containing cells. In this study, the role of caspases in hydroquinone-induced apoptosis in MPO-rich HL-60 promyelocytic leukemia and MPO-deficient Jurkat T-lymphoblastic leukemia cells was investigated. HQ-induced apoptosis in both cell types was accompanied by phosphatidylserine (PS) exposure, caspases-3/-7 activation, PARP cleavage, DNA fragmentation, and ultrastructural changes as assessed by electron microscopy. In HL-60 cells, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) blocked activation of caspases-3/-7, cleavage of PARP, and DNA, but PS externalization and cytoplasmic changes were not significantly affected. In marked contrast, all features of apoptosis were completely inhibited by Z-VAD.FMK in HQ-treated Jurkat cells. These data provide evidence for Z-VAD.FMK-insensitive and caspases-3/-7-independent pathway(s) in the externalization of PS and cytoplasmic changes during HQ-induced apoptosis in HL-60 cells. In contrast, in Jurkat cells, all of these changes required caspase activation. The ability of HQ to induce equivalent apoptosis in both MPO-deficient Jurkat cells and MPO-rich HL-60 cells demonstrates that MPO-catalyzed bioactivation of HQ is not a prerequisite for toxicity. The differential mechanisms of apoptosis in HL-60 and Jurkat T cells may reflect the MPO activity of these cells and, as a result, the amount of reactive BQ and other metabolites that are generated.

Relationship between NAD(P)H:quinone oxidoreductase 1 (NQO1) levels in a series of stably transfected cell lines and susceptibility to antitumor quinones.

To investigate the importance of NAD(P)H:quinone oxidoreductase 1 (or DT-diaphorase; NQO1) in the bioactivation of antitumor quinones, we established a series of stably transfected cell lines derived from BE human colon adenocarcinoma cells. BE cells have no NQO1 activity due to a genetic polymorphism. The new cell lines, BE-NQ, stably express wild-type NQO1. BE-NQ7 cells expressed the highest level of NQO1 and were more susceptible [determined by the thiazolyl blue (MTT) assay] to known antitumor quinones and newer clinical candidates. Inhibition of NQO1 by pretreatment with an irreversible inhibitor, ES936 [5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione], protected BE-NQ7 cells from toxicity induced by streptonigrin, ES921 [5-(aziridin-1-yl)-3-(hydroxymethyl)-1,2-dimethylindole-4,7-dione], and RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone]. RH1 was evaluated further by clonogenic assay for cytotoxic response and was more cytotoxic to BE-NQ7 cells than to BE cells. Cytotoxicity was abrogated by inhibition of NQO1 with ES936 pretreatment. Using a comet assay to evaluate DNA cross-linking, BE-NQ7 cells demonstrated significantly higher DNA cross-links than did BE cells in response to RH1 treatment. DNA cross-linking in BE-NQ7 cells was observed at very low concentrations of RH1 (5 nM), confirming that NQO1 activates RH1 to a potent cross-linking species. Further studies using streptonigrin, ES921, and RH1 were undertaken to analyze the relationship between NQO1 activity and quinone toxicity. Toxicity of these compounds was measured in a panel of BE-NQ cells expressing a range of NQO1 activity (23-433 nmol/min/mg). Data obtained suggest a threshold for NQO1-induced toxicity above 23 nmol/min/mg and a sharp dose-response curve between the no effect level of NQO1 (23 nmol/min/mg) and the maximal effect level (>77 nmol/min/mg). These data provide evidence that NQO1 can bioactivate antitumor quinones in this system and suggest that a threshold level of NQO1 activity is required to initiate toxic events.

Rapid polyubiquitination and proteasomal degradation of a mutant form of NAD(P)H:quinone oxidoreductase 1.

The NAD(P)H:quinone oxidoreductase 1 (NQO1)*2 polymorphism is characterized by a single proline-to-serine amino acid substitution. Cell lines and tissues from organisms genotyped as homozygous for the NQO1*2 polymorphism are deficient in NQO1 activity. In studies with cells homozygous for the wild-type allele and cells homozygous for the mutant NQO1*2 allele, no difference in the half-life of NQO1 mRNA transcripts was observed. Similarly, in vitro transcription/translation studies showed that both wild-type and mutant NQO1 coding regions were transcribed and translated into full-length protein with equal efficiency. Protein turnover studies in NQO1 wild-type and mutant cell lines demonstrated that the half-life of wild-type NQO1 was greater than 18 h, whereas the half-life of mutant NQO1 was 1.2 h. Incubation of NQO1 mutant cell lines with proteasome inhibitors increased the amount of immunoreactive NQO1 protein, suggesting that mutant protein may be degraded via the proteasome pathway. Additional studies were performed using purified recombinant NQO1 wild-type and mutant proteins incubated in a rabbit reticulocyte lysate system. In these studies, no degradation of wild-type NQO1 protein was observed; however, mutant NQO1 protein was completely degraded in 2 h. Degradation of mutant NQO1 was inhibited by proteasome inhibitors and was ATP-dependent. Mutant NQO1 incubated in rabbit reticulocyte lysate with MG132 resulted in the accumulation of proteins with increased molecular masses that were immunoreactive for both NQO1 and ubiquitin. These data suggest that wild-type NQO1 persists in cells whereas mutant NQO1 is rapidly degraded via ubiquitination and proteasome degradation.

Effects of parenteral cysteine and glutathione feeding in a baboon model of severe prematurity.

BACKGROUND: The availability of cysteine for glutathione synthesis is low in premature infants with respiratory distress. OBJECTIVE: The effects of gestational age, oxygen delivery, and cysteine infusion or glutathione infusion, or both, on plasma total cysteine and other methionine metabolites were studied in a baboon model of severe premature birth with respiratory distress. DESIGN: Premature baboons were studied as part of the multiinvestigator National Institutes of Health Collaborative Project on Bronchopulmonary Dysplasia. Premature baboons, 125 d (69% of term) or 140 d (78% of term) of gestational age, were maintained in neonatal intensive care units for

NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase that is involved in chemoprotection and can also bioactivate certain antitumor quinones. This review focuses on detoxification reactions catalyzed by NQO1 and its role in antioxidant defense via the generation of antioxidant forms of ubiquinone and vitamin E. Bioactivation reactions catalyzed by NQO1 are also summarized and the development of new antitumor agents for the therapy of solid tumors with marked NQO1 content is reviewed. NQO1 gene regulation and the role of the antioxidant response element and the xenobiotic response element in transcriptional regulation is summarized. An overview of genetic polymorphisms in NQO1 is presented and biological significance for chemoprotection, cancer susceptibility and antitumor drug action is discussed.

Genotype-phenotype relationships in studies of a polymorphism in NAD(P)H:quinone oxidoreductase 1.

The NAD(P)H:quinone oxidoreductase 1 (NQO1) genotype-phenotype relationship was examined in individuals with a polymorphism in NQO1. The polymorphism comprises a C to T base change at position 609 of the human NQO1 cDNA (C609T) and codes for a proline to serine substitution in the amino acid structure of the NQO1 protein. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism analysis of genomic DNA. Phenotyping was performed using enzyme activity assays and/or immunoblotting of human tumor cell lines and of saliva and bone marrow samples from healthy donors. Phenotyping of uninvolved lung and lung tumors from archived biopsy material was performed by immunohistochemistry. NQO1 activity and protein could be detected in wild-type (C/C) human tumor cells (HT-29) under conditions where NQO1 protein could not be detected in cells (BE) homozygous for the C609T change (T/T). Trace levels of NQO1 protein could be detected in BE cells; however, when immunoblots were subjected to chemiluminescence detection for prolonged periods. In saliva samples from 11 individuals carrying the homozygous C609T change (T/T), no NQO1 protein could be detected even after prolonged chemiluminescence detection. The amount of NQO1 protein present in saliva was quantified and found to be significantly less in heterozygous individuals (C/T) than in wild-type individuals (C/C). In bone marrow stromal cultures, both NQO1 activity and protein could be detected in heterozygotes (C/T) and in wild-type (C/C) samples. In a bone marrow stromal culture from an individual genotyped as T/T at position 609, no NQO1 protein or activity could be detected. NQO1 is elevated in non-small cell lung cancers and could be readily observed as intense immunostaining throughout lung adenocarcinomas genotyped as C/C but no immunostaining could be detected in adenocarcinomas genotyped as T/T at position 609. NQO1 is expressed in normal human lung but is localized to respiratory epithelium and to vascular endothelium. In normal lung tissue from individuals genotyped as T/T, no or faint immunostaining for NQO1 could be detected in either respiratory epithelium or vascular endothelium. These results demonstrate that tissues from individuals homozygous for the C609T change have no detectable or, at best, only trace amounts of NQO1 protein and are devoid of NQO1 activity.

A new screening system for NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones: identification of a new aziridinylbenzoquinone, RH1, as a NQO1-directed antitumor agent.

NAD(P)H:quinone oxidoreductase (NQO1; DT-diaphorase) is elevated in certain tumors, such as non-small cell lung cancer (NSCLC). Compounds such as mitomycin C and streptonigrin are efficiently bioactivated by NQO1 and have been used in an enzyme-directed approach to chemotherapy. Previously, 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone (MeDZQ) was identified as a potential antitumor agent based on its high rate of bioactivation by human NQO1 and its selective cytotoxicity to cells containing elevated NQO1. RH1, a water-soluble analogue of MeDZQ synthesized in this work, was a better substrate for recombinant human NQO1 than the parent compound. RH1 was, correspondingly, more cytotoxic to human tumor cells expressing elevated NQO1 activity (H460 NSCLC and HT29 human colon carcinoma), as measured by 3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium assay, than it was to cells deficient in NQO1 activity (H596 NSCLC and BE human colon carcinoma). RH1 exhibited a greater selective toxicity (ratio of IC50s in H596:H460 and BE:HT29) to cells with elevated NQO1 activity relative to MeDZQ. Additionally, we report the establishment of a stable line of BE human colon carcinoma cells transfected with wild-type human NQO1 (BE-NQ7). BE cells are devoid of NQO1 activity due to a homozygous point mutation in the NQO1 gene. In comparison to the parental cell line, RH1, MeDZQ, and mitomycin C were significantly more cytotoxic to BE-NQ7 cells (17-, 7-, and 3-fold, respectively), confirming that the presence of NQO1 is sufficient to increase cytotoxicity of these antitumor quinones. These data suggest that RH1 may be an effective NQO1-directed antitumor agent for the therapy of tumors with elevated NQO1 activity, such as NSCLC.

Arsenate toxicity in human erythrocytes: characterization of morphologic changes and determination of the mechanism of damage.

Chronic arsenic exposure is associated with alterations in peripheral circulation and vascular disease. Toxicity to the vasculature is documented, but the effect of arsenic on the erythrocyte has not been evaluated. To determine if arsenic was toxic to human erythrocytes and whether this could contribute to vascular disease, human erythrocytes were incubated in vitro with sodium arsenate, As(V), or sodium arsenite, As(III), and assessed for damage. After 5 h of incubation with 10 mM As(V) or As(III), significant cell death (hemolysis) only occurred in the As(V) treated cells. Morphologic changes were assessed by scanning electron microscopy and light microscopy. As(V) induced a classic discocyte-echinocyte transformation extending to the formation of sphero-echinocytes; these changes were concentration dependent. As(III) treatment also resulted in echinocyte formation but less extensive than in As(V) treated cells, and no sphero-echinocytes were formed. The observed damage was consistent with reported changes induced by ATP depletion, and measurement of ATP in these samples confirmed this as a mechanism of damage. As(V) treatment at concentrations as low as 0.01 mM for 5 h significantly depleted ATP, and As(III) was relatively ineffective in causing ATP depletion. Based on these three parameters, the erythrocyte was estimated to be as much as 1000 times more susceptible to As(V) than As(III). ATP is required for the cell to maintain membrane integrity and deform efficiently in circulation. The changes described here could contribute to vascular occlusion, ischemia, and tissue death associated with arsenic circulatory disorders.

Sequence of toxic events in arsine-induced hemolysis in vitro: implications for the mechanism of toxicity in human erythrocytes.

Arsine, the hydride of arsenic (AsH3), is the most acutely toxic form of arsenic, causing rapid and severe hemolysis upon exposure. The mechanism of action is not known, and there are few detailed investigations of the toxicity in a controlled system. To examine arsine hemolysis and understand the importance of various toxic responses, human erythrocytes were incubated with arsine in vitro, and markers of toxicity were determined as a function of time. The earliest indicators of damage were changes in sodium and potassium levels. Within 5 min incubation with 1 mm arsine, the cells lost volume control, manifested by leakage of potassium, influx of sodium, and increases in hematocrit. Arsine did not, however, significantly alter ATP levels nor inhibit ATPases. These changes were followed by profound disturbances in membrane ultrastructure (examined by light and electron microscopy). By 10 min, significant numbers of damaged cells formed, and their numbers increased over time. These events preceded hemolysis, which was not significant until 30 min. It has been proposed that arsine interacts with hemoglobin to form toxic hemoglobin oxidation products, and this was also investigated as a potential cause of hemolysis. Essentially on contact with arsine, methemoglobin was formed but only reached 2-3% of the total cellular hemoglobin and remained unchanged for up to 90 min. There was no evidence that further oxidation products (hemin and Heinz bodies) were formed in this system. Based on these observations, hemolysis appears to be dependent on membrane disruption by a mechanism other than hemoglobin oxidation.

Interactions of rat red blood cell sulfhydryls with arsenate and arsenite.

Arsenic-thiol interactions were investigated by determining changes in rat blood sulfhydryls after exposure to arsenate, As(V), or arsenite, As(III). Incubation with As(V) resulted in time- and dose-dependent depletion of nonprotein sulfhydryls (NPSH), specifically glutathione (GSH). At the highest As(V) concentration (10 mM), significant loss of glutathione was only observed after 3 h of incubation, but by 5 h 0.5 mM As(V) and higher was sufficient to deplete GSH. As(V) was reduced to As(III) at all dose levels, indicating a redox interaction with GSH, but oxidized glutathione (GSSG) was not formed in sufficient quantities to account for losses in GSH. This may be due to formation of another oxidized species such as a protein-mixed-disulfide (ProSSG). Further evidence that glutathione reduces arsenate was obtained by pretreating cells with the sulfhydryl derivatizing agent N-ethylmaleimide (NEM). Removal of thiols with NEM severely inhibited the formation of As(III) in these incubations, indicating that the main pathway for arsenate reduction in red cells is sulfhydryl dependent. As(III) demonstrated a completely different profile of sulfhydryl interaction. Sulfhydryls (NPSH and GSH) were depleted but the losses were primarily accounted for by oxidation to GSSG. As(III) was also a more potent sulfhydryl depleting agent, requiring only 0.1 mM As(III) to significantly reduce GSH after 5 h of incubation. Significant levels of GSSG formed at all doses of As(III). Evidence is presented to suggest that As(III) also formed mixed complexes with protein and glutathione. Samples that were acid precipitated displayed loss of cytosolic glutathione, which could be reversed if NEM was added prior to protein precipitation. Arsenic was detected in high quantities in the protein precipitates, and this was also found to be reversible by NEM treatment. The fact that both GSH depletion and protein binding were reversible by NEM treatment points to formation of a mixed complex of protein, GSH, and As(III), possibly ProS-As-(SG)x. Arsenic affinity chromatography and polyacrylamide gel electrophoresis were used to characterize arsenic binding proteins in red-cell cytosol. The main arsenic binding protein appeared to be hemoglobin.